The discovery of new or divergent viruses using metagenomics and high-throughput sequencing has become more commonplace. The preparation of a sample is known to have an effect on the representation of virus sequences within the metagenomic dataset yet comparatively little attention has been given to this. Physical enrichment techniques are often applied to samples to increase the number of viral sequences and therefore enhance the probability of detection. With the exception of virus ecology studies, there is a paucity of information available to researchers on the type of sample preparation required for a viral metagenomic study that seeks to identify an aetiological virus in an animal or human diagnostic sample. A review of published virus discovery studies revealed the most commonly used enrichment methods, that were usually quick and simple to implement, namely low-speed centrifugation, filtration, nuclease-treatment (or combinations of these) which have been routinely used but often without justification. These were applied to a simple and well-characterised artificial sample composed of bacterial and human cells, as well as DNA (adenovirus) and RNA viruses (influenza A and human enterovirus), being either non-enveloped capsid or enveloped viruses. The effect of the enrichment method was assessed by both quantitative real-time PCR and metagenomic analysis that incorporated an amplification step. Reductions in the absolute quantities of bacteria and human cells were observed for each method as determined by qPCR, but the relative abundance of viral sequences in the metagenomic dataset remained largely unchanged. A 3-step method of centrifugation, filtration and nuclease-treatment showed the greatest increase in the proportion of viral sequences. This study provides a starting point for the selection of a purification method in future virus discovery studies, and highlights the need for more data to validate the effect of enrichment methods on different sample types, amplification, bioinformatics approaches and sequencing platforms. This study also highlights the potential risks that may attend selection of a virus enrichment method without any consideration for the sample type being investigated.

Influenza epidemics and pandemics have significant impacts on economies, morbidity and mortality worldwide. The ability to rapidly and accurately sequence influenza viruses is instrumental in the prevention and mitigation of influenza. All eight influenza genes from an influenza A virus were amplified by PCR simultaneously and then subjected to sequencing on a MinION nanopore sequencer. A complete influenza virus genome was obtained that shared greater than 99% identity with sequence data obtained from Illumina MiSeq and traditional Sanger-sequencing. The laboratory infrastructure and computing resources used to perform this experiment on the MinION nanopore sequencer would be available in most molecular laboratories around the world. Using this system, the concept of portability, and thus sequencing influenza viruses in the clinic or field is now tenable.

There is much interest in using high-throughput DNA sequencing methodology to monitor microorganisms, complex plant and animal communities. However, there are experimental and analytical issues to consider before applying a sequencing technology, which was originally developed for genome projects, to ecological projects. Many of these issues have been highlighted by recent microbial studies. Understanding how high-throughput sequencing is best implemented is important for the interpretation of recent results and the success of future applications. Addressing complex biological questions with metagenomics requires the interaction of researchers who bring different skill sets to problem solving. Educators can help by nurturing a collaborative interdisciplinary approach to genome science, which is essential for effective problem solving. Educators are in a position to help students, teachers, the public and policy makers interpret the new knowledge that metagenomics brings. To do this, they need to understand, not only the excitement of the science but also the pitfalls and shortcomings of methodology and research designs. We review these issues and some of the research directions that are helping to move the field forward.

Wild birds harbor a huge diversity of avian avulaviruses (formerly avian paramyxoviruses). Antarctic penguin species have been screened for avian avulaviruses since the 1980s and, as such, are known hosts of these viruses. In this study, we screened three penguin species from the South Shetland Islands and the Antarctic Peninsula for avian avulaviruses. We show that Adelie penguins (Pygoscelis adeliae) are hosts for four different avian avulavirus species, the recently described avian avulaviruses 17 to 19 and avian avulavirus 10-like, never before isolated in Antarctica. A total of 24 viruses were isolated and sequenced; avian avulavirus 17 was the most common, and phylogenetic analysis demonstrated patterns of occurrence, with different genetic clusters corresponding to penguin age and location. Following infection in specific-pathogen-free (SPF) chickens, all four avian avulavirus species were shed from the oral cavity for up to 7 days postinfection. There was limited shedding from the cloaca in a proportion of infected chickens, and all but one bird seroconverted by day 21. No clinical signs were observed. Taken together, we propose that penguin species, including Antarctic penguins, may be the central reservoir for a diversity of avian avulavirus species and that these viruses have the potential to infect other avian hosts. IMPORTANCE Approximately 99% of all viruses are still to be described, and in our changing world, any one of these unknown viruses could potentially expand their host range and cause epidemic disease in wildlife, agricultural animals, or humans. Avian avulavirus 1 causes outbreaks in wild birds and poultry and is thus well described. However, for many avulavirus species, only a single specimen has been described, and their viral ecology and epidemiology are unknown. Through the detection of avian avulaviruses in penguins from Antarctica, we have been able to expand upon our understanding of three avian avulavirus species (avian avulaviruses 17 to 19) and report a potentially novel avulavirus species. Importantly, we show that penguins appear to play a key role in the epidemiology of avian avulaviruses, and we encourage additional sampling of this avian group.

Incidence of human yersiniosis in New Zealand has increased between 2013 and 2017. For surveillance and outbreak investigations it is essential that an appropriate level of discrimination between pathogenicYersinia enterocoliticaisolates is provided, in order to support epidemiological linking of connected cases. Subtyping of 227Y. enterocoliticaisolates was performed using a range of different typing methods, including biotyping, serotyping and seven loci multiple-locus variable-number tandem-repeat analysis (MLVA). In addition, core genome single-nucleotide polymorphism (core SNP) analysis and multi-locus sequence typing were performed on a subset of 69 isolates. Sixty-seven different MLVA types were identified. One MLVA profile was associated with an outbreak in the Bay of Plenty region, supported by epidemiological data. Core SNP analysis showed that all the outbreak-related isolates clustered together. The subtyping and epidemiological evidence suggests that the outbreak of yersiniosis in the Bay of Plenty region between October and December 2016 could be attributed to a point source. However, subtyping results further suggest that the same clone was isolated from several regions between August 2016 and March 2017. Core SNP analysis and MLVA typing failed to differentiate betweenY. enterocoliticabiotype 2 and biotype 3. For this reason, we propose that these biotypes should be reported as a single type namely:Y. enterocoliticabiotype 2/3 and that the serotype should be prioritised as an indicator of prevalence.

PREMISE OF THE STUDY: Despite attempts to degrade the sporopollenin in pollen walls, this material has withstood a hundred years of experimental treatments and thousands of years of environmental attack in insects and soil. We present evidence that sporopollenin, nonetheless, locally degrades only minutes after pollination in Arabidopsis thaliana flowers, and describe here a two‐part pollen germination mechanism in A. thaliana involving both chemical weakening of the exine wall and swelling of the underlying intine.METHODS: We explored naturally occurring components from pollen and stigma surfaces and found a tripartite mix of hydrogen peroxide, peroxidase and catalase enzymes (all at high levels at the pollination interface) to be experimentally sufficient to degrade the sporopollenin of some Brassicaceae family members.KEY RESULTS: At pollination, factors carried on the pollen surface may mix with factors on the stigma surface in a reaction that locally oxidizes the exine pollen wall. Hydrogen peroxide, catalases, and peroxidases are biologically present at the right time and place and, when mixed experimentally, are sufficient to degrade the walls of susceptible pollen.CONCLUSIONS: Our work on native biochemistry for breaching sporopollenin suggests new research directions in pollen aperture evolution and could aid efforts to analyze sporopollenin's composition, needed for application of this corrosion‐resistant, but long‐intractable material.